The present invention relates to a method for the enantioselective reduction of 3,5-dioxo-carboxylic acids, their salts and their esters.
Homochiral 3,5-dihydroxycarboxylic acid derivatives having Formula 1 are intermediates in the synthesis of numerous natural and active substances. 
wherein X stands for a component from the group consisting of hydrogen, halogen, alkyl, aryl, CHxe2x95x90CHR2, Cxe2x89xa1CR3, (wherein R2=R, except for metal cation, and R3xe2x95x90R); xcexa3 stands for H or for a protective group for the hydroxyl function; R stands for H, metal cation, for an alkyl, aryl, aralkyl or cycloalkyl radical.
Depending on the absolute configuration at the stereo centers C-3 and C-5, they can be systematically employed in the synthesis of chiral natural substances, such as mevic acids, or synthetic HMG-CoA-reductase inhibitors.
Other natural or active substances call for different configurations of the stereogenic centers in position C-3 and C-5. Consequently, there is great interest in the preparation of all possible stereoisomers of 3,5-dihydroxycarboxylic acid derivatives according to Formula 1 in an optically pure form. An advantageous method for the preparation of these compounds is the catalytic enantioselective reduction of the prochiral 3,5-dioxocarboxylic acid derivatives according to Formula 2. 
When this method is employed, there is no need for costly and environmentally burdensome separation procedures for the racemate, scalemate or diastereomer. This avoids the binding and cleavage of a stoichiometric quantity of a homochiral auxiliary group that are necessary in a diastereoselective synthesis. Moreover, the carbon skeleton of the 3,5-di-hydroxycarboxylic acid ester according to Formula 1 is already complete in the initial compounds according to Formula 2, that is to say, the stereocenters are only introduced into the overall synthesis sequence at a later point in time, as a result of which the loss of homochiral material is kept low.
European Patent Application No. 0,569,998 A2 discloses an enantioselective microbial process to reduce di-ketoesters that are oxyalkyl-substituted and oxyaralkyl-substituted in position 6.
WO 97/00968 discloses a process to reduce 3-oxo-5-hydroxy-carboxylic acid esters by means of reductases of Beauveria, Candida, Kluyveromycis, Torulaspora or Pichia.
DE 196 10 984 A1 discloses a stable microbial enzyme with alcohol-dehydrogenase activity, a process to obtain it as well as its use for the enantioselective reduction/oxidation of organic keto compounds/hydroxy compounds whereby, depending on the type of initial compounds, either R-hydroxy or S-hydroxy compounds are obtained.
The publication titled xe2x80x9cEnantioselective microbial reduction of 3,5-dioxo-6-(benzyloxyl)hexanoic acid ethyl esterxe2x80x9d in Enzyme Microb. Technol. (1993), 15 (12), 1014-21 ff. shows the reduction of 3,5-dioxo-6-(benzyloxy)hexanoic acid ethyl ester by means of a reductase.
As far as the terminology is concerned, a definition of the term will be introduced here as a working term that is to be valid within the disclosure: 
In Formula 3, the OH group projects from the paper plane in position 5, whereas the chains with the carboxylic acid function or with the ester function as well as with the radical X lie in the paper plane. Therefore, the hydrogen atom on C-5 recedes behind the paper plane. In accordance with the CIP (RS) nomenclature, as a function of the priority of the chain that is substituted with X, an R-designation or an S-designation would be employed for the same spatial configuration of the OH group projecting from the paper plane towards the front. As defined by the invention, the designation r-configuration will be used for an arrangement of the substituent used in which the OH group in position 5 projects forward from the paper plane, in which the hydrogen atom in position 5 extends below the paper plane and in which the side chain provided with the carboxylic acid function or with the ester function lies on the right-hand side of the fifth carbon atom while the side chain provided with the substituent X lies on the left-hand side of the fifth carbon atom. For those cases where the right-hand side chain provided with the carboxylic acid function or with the ester function has a higher priority than the left-hand side chain provided with the substituent X, this corresponds to the classic R-configuration. If the priorities of the above-mentioned chains are reversed (for example, by selecting X=halogen), then the classic S-configuration would be ascribed to the target compound. Both cases, however, should be encompassed by the definition of an r-configuration.
The objective of the invention is to regioselectively introduce an r-configuration of the hydroxyl group in position 5 during the enzymatic reduction of 3,5-dioxocarboxylic acid derivatives.
Another object of the invention is to provide a new method for preparing enantiomerpure 3,5-dihydroxycarboxylic acid derivatives, in other words, to create a process with which a syn-reduction or an anti-reduction of the keto group can systematically take place in position 3 towards position 5, so that the 3,5-dihydroxycarboxylic acid derivative has a tailor-made, absolute configuration with respect to C-3 which can be prepared as desired.
Another objective of the invention is to create an improved method for the synthesis of 3,5-dioxocarboxylic acid esters according to Formula 4 that can be used as educts for the enzymatic reduction.
On the basis of the generic part of claim 1, the objective is achieved according to the invention by means of the features indicated in the characterizing part of claim 1.
With the method according to the invention, it is now possible to reduce 3,5-dioxo carboxylic acids as well as their esters in a highly enantioselective manner and thus to obtain compounds that can then be employed in a synthesis of natural and active substances that have a defined absolute configuration in the 3,5-dihydroxycarboxylic acid structural element.
Advantageous embodiments of the invention are described in the subordinate claims.
The invention will be described below in a general manner.
According to the invention, a compound according to Formula 4
wherein R1 stands for a component from the group consisting of alkyl, alkenyl, cyclo-alkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl, hydrogen or metal cation, and X stands for a component from the group consisting of hydrogen, halogen, alkyl, aryl, CHxe2x95x90CHR2, Cxe2x89xa1CR3 (wherein R2xe2x95x90R1, except for metal cation, and R3xe2x95x90R1) is reacted by means of an alcohol dehydrogenase with the addition of NADPH or of another co-factor.
The term alkyl refers to straight-chain as well as to branched saturated carbon chains. Examples of these are methyl, ethyl, n-propyl, i-propyl, t-butyl, pentyl, i-pentyl, n-hexyl, i-hexyl. The term alkenyl relates to straight-chain and branched unsaturated hydro-carbons, examples of which are vinyl, 1-propenyl, allyl, butenyl, i-butenyl. The term cycloalkyl encompasses saturated, ring-shaped hydrocarbon chains consisting of three, four, five, six or seven carbon atoms. Cycloalkenyl designates unsaturated, ring-shaped hydrocarbons having 5, 6, 7 or 8 carbon atoms. Aryl refers to aromatic systems, enclosed heteroaromatic compounds and substituted aromatic systems, such as phenyl, p-tolyl, furanyl. Aralkyl refers to aryl radicals that are bonded via alkyl groups such as, for instance, a benzyl radical. The term cycloalkylalkyl comprises cycloalkyl radicals that are bonded via alkyl groups. Halogen preferably refers to fluorine and chlorine.
The alcohol dehydrogenase is preferably recombinant and stems from Lactobacillus, especially Lactobacillus brevis (recLBADH). The particularly advantageous aspect of this enzyme is that it can be recombinantly over-expressed and thus can be made available in large quantities. This also allows its use on a large, technical scale. The reaction can take place in an aqueous medium by means of an enzyme as well as on the intracellular level in a microorganism. In a preferred embodiment, recLBADH from Lactobacillus brevis which was recombinantly over-expressed in Escherichia coli is used. Another advantage of recLBADH is the capability of the enzyme to regenerate the necessary co-factor NADPH in a substrate-coupled manner, that is to say, NADPH does not have to be employed in a stoichiometric amount. Moreover, this avoids the need to add a second enzyme for purposes of regenerating the NADPH, which ultimately also reduces costs. With this transfer hydrogenation, an alcohol, preferably isopropanol, can serve as the hydrogen donor. The activity of recLBADH wan be increased by adding Mg2+.
The method according to the invention can be carried out at room temperature since the enzyme recLBADH advantageously has a high heat stability. This also means that less enzyme is needed for a given yield, which translates into cost savings. Complex cooling measures can likewise be dispensed with. The method according to the invention, however, can also be conducted at the temperatures commonly employed for enzymatic reactions, between 0xc2x0 C. and 70xc2x0 C. [32xc2x0 F. and 158xc2x0 F.]. Preference is given to the range between 20xc2x0 C. and 50xc2x0 C. [68xc2x0 F. and 122xc2x0 F.].
The method according to the invention can be carried out at a pH value of 5.5, since the preferred enzyme displays a stability maximum at a pH of 5.5. This is advantageous since, at this pH value, many ester substrates according to Formula 4 exhibit a higher stability than at the higher pH values which normally have to be maintained in enzymatic reactions. The reaction, however, can also be carried out within a pH range from 5.5 to 9, preferably in the range between 5.5 and 6.5.
In order to ensure a suitable pH value, any buffer substance suitable for an enzymatic reaction can be used. Examples of these are triethanol amine (TEA), phosphate buffer or TRIS buffer. The concentration ranges for the buffers advantageously lie between 50 and 500 mmol/L.
The reaction product with the r-configuration is depicted in Formula 5, wherein the substituents R1 and X have the same meaning as in Formula 4. 
The enantiomer surpluses for these reactions can be said to be xe2x89xa698% to 100%.
In a refinement of the invention, the keto group of the compound in position 3 in Formula 5 is diastereoselectively reduced to form an OH group which is then in the syn-position or anti-position relative to the OH group in position 5.
The compound according to Formula 5 can be reacted to form the reaction product 6a, 6b employing methods that are known for the synthesis of syn-diols (6a) and anti-diols (6b). 
For the production of syn-diols (6a) from xcex2-hydroxy carbonyl compounds, these are, for example, sodium boron hydride reduction in the presence of trialkyl boranes or alkoxy-dialkyl boranes ([1] K. Narasaka, F. C. Pai, Tetrahedron 1984, 40, 2233-2238; [2] K. M. Chen, G. E. Hardtmann, K. Prasad, O. Repic, M. J. Shapiro, Tetrahedron Lett. 1987, 28, 155-158) while for the production of anti-diols (6b) from xcex2-hydroxy carbonyl compounds, these are for example, reduction with tetramethyl ammonium triacetoxy boron hydride (D. A. Evans, K. T. Chapman, E. M. Carreira, J. Am. Chem. Soc. 1988, 110, 3560-3578).
Generally speaking, both chemical and enzymatic reductions are possible in the second step. The enzymatic reduction of position 3 can be carried out, for instance, with the following microorganisms or their isolated reductases: Beauveria bassiana ATCC 7159, Candida humicola CBS 1897, Candida diddensiae ATCC 20213, Candida frieddrichii ATCC 22970, Candida solani CBS 1908, Hansenula nonfementans CBS 5764, Kluyveromyces drosophilarum CBS 2105, Pichia angusta NCYC 495, Pichia angusta NCYC R320, Pichia angusta NCYC R322, Pichia haplo-phila CBS 2028, Pichia membranefaciens DSM 70366, Pichia pastoris BPCC 260, Pichia pastoris BPCC 443, Pichia pastoris NCYC R321, Torulaspora hansenii ATCC 20220, Candida pelliculosa ATCC 2149, Hansenula anomola CBS 2230, Neurospora crassa ATCC 9277, Pichia trehalophila CBS 5361, Mortierella alpina MF 5534 (ATCC8979).
Other options are also the reductases from Saccharomyces, especially Saccharomyces cerevisiae, both in the cell as well as in isolated form.
The production method according to the invention can be advantageously carried out in a continuous process in an enzyme membrane reactor, as described, for example, in German Patent No. 39 37 892.
The enantioselective reduction of 3,5-dioxocarboxylic acid esters at position C-5 can take place with wild type enzymes and/or recombinant over-expressed enzymes as well as with whole cells. Preference, however, is given to extracellular reaction with a cell raw extract, since higher enantiomer surpluses are achieved.
In an advantageous embodiment of the invention, the substrate needed for the synthesis is prepared according to a method which, in contrast to other well-established methods for the synthesis of 3,5-dioxocarboxylic acid derivatives according to Formula 4, can make do with particularly inexpensive and simple initial materials. Another advantage of this method is its uncomplicated reaction engineering.
The method according to the invention will be described below by means of which high yields of 3,5-dioxocarboxylic acid esters according to Formula 2 can be obtained by acylating bisenolates according to Formula A, wherein R4 stands for alkyl, alkenyl, cyclo-alkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl or metal cation 
with easily available and inexpensive carboxylic acid esters according to Formula B, wherein X has the same meaning as in Formula 2 and wherein R5 stands for an alkyl radical. 
According to known work methods, a bisenolate according to Formula A is made in situ on the basis of a xcex2-ketoester with a lithium compound in an organic solvent. While the internal temperature of the reaction vessel is controlled, this bisenolate according to Formula A is then acylated by the addition of a carboxylic acid ester according to Formula B, whereby preference is given to the use of xcex1-halogen carboxylic acid ester. Particularly preferred are the methyl esters of chloroacetic acid and of fluoroacetic acid. Subsequently, the mixture undergoes an acidic aqueous treatment in the presence of an organic solvent that is not miscible with water such as, for instance, acetic acid ethyl ester or diethyl ether.
The reaction according to the invention is preferably carried out in an inert-gas atmosphere under anhydrous conditions. An example of an inert gas is nitrogen or argon. Suitable solvents are inert organic solvents such as ethers, alkanes or cycloalkanes (C5-C7), toluene or benzene. Preference is given to aprotic coordinating solvents from the compound class of the ethers such as, for example, diethyl ether, dimethoxyethane or tetrahydrofuran (THF). THF is especially preferred.
The lithium compound is preferably highly alkaline, but not very nucleophilic. Preferred are lithium amides such as, for instance, lithium diisopropyl amide (LDA), lithium dicyclohexyl amide, lithium cyclohexyl isopropyl amide, lithium 2,2,6,6-tetramethyl piperidine (LiTMP) or lithium-bis-(trimethylsilyl)-amide (LiHMDS). LDA is particularly preferred. Other possibilities are organo-lithium compounds such as, for example, mesityl lithium or t-butyl lithium. The preferred molar ratio of lithium base to xcex2-ketoester in order to produce the bisenolate ranges from 2:1 to 4:1, a molar ratio of 2.1:1 being particularly preferred.
The acylation of the bisenolate according to Formula B created in situ by means of the addition of a carboxylic acid ester according to Formula B is preferably done at an internal temperature of the reaction vessel ranging from xe2x88x92100xc2x0 C. to +25xc2x0 C. [xe2x88x92148xc2x0 F. to +77xc2x0 F.], at best between xe2x88x9280xc2x0 C. and xe2x88x9240xc2x0 C. [xe2x88x92112xc2x0 F. to xe2x88x9240xc2x0 F.]. Special preference is given to the range between xe2x88x9272xc2x0 F. and xe2x88x9265xc2x0 C. [xe2x88x9297.6xc2x0 F. to xe2x88x9285xc2x0 F.]. The molar ratio of carboxylic acid ester to bisenolate preferably lies between 0.5:1 and 2:1. Especially preferred is a ratio of 1:1.
The preparation of the highly alkaline reaction mixture preferably takes place two to 120 minutes after the addition of the last portion of carboxylic acid ester according to Formula B, at best after 15 to 30 minutes. For this purpose, the contents of the reaction vessel are poured into a cooled and vigorously stirred mixture consisting of an organic solvent that is not miscible with water, for example, acetic acid ethyl ester or diethyl ether, and an aqueous solution of an acid such as, for instance, diluted hydrochloric acid, acetic acid or ammonium chloride solution. In this context, preference is given to the use of bimolar hydrochloric acid and acetic acid ethyl ester.
In conjunction with the acylation of bisenolates according to Formula A with carboxylic acid esters according to Formula B, this approach constitutes an improvement over existing methods since a surplus of base or bisenolate is avoided and so is the use of special, expensive acylation reagents, catalysts or co-solvents. ([a] M. Yamaguchi, K. Shinato, H. Nakashima, T. Minami, Tetrahedron 1988, 44, 4767-4775; [b] N. S. Narasimhan, R. K. Ammanamanchi, J. Org. Chem. 1983, 48, 3945-3947; [c] S. N. Huckin, L. Weiler, Can. J. Chem. 1974, 52, 1343-1351). Another advantage is the uncomplicated reaction engineering since, unlike in Huckin et al., it is not necessary to add the components at alternating intervals, but rather, the necessary volumes can be added all at once. With the method according to the invention, no significant amount of by-products is formed. Various 3,5-dioxocarboxylic acid esters according to Formula 2 were prepared using the method according to the invention, examples of which are the compounds 6-chloro-3,5-dioxohexanoic acid-1,1-dimethyl ethyl ester and 6-fluoro-3,5-dioxohexanoic acid-1,1-dimethyl ethyl ester. Both compounds are new.